CN105646684A - Rice grain shape related protein GLW2 and encoding gene and application thereof - Google Patents
Rice grain shape related protein GLW2 and encoding gene and application thereof Download PDFInfo
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Abstract
The invention provides rice grain shape related protein GLW2 and an encoding gene and application thereof. The protein GLW2 has an amino acid sequence shown as in SEQ ID No. 2 or an equivalently functional amino acid sequence formed by subjecting an amino acid sequence shown in SEQ ID No. 2 to substitution, deletion or addition via one or more amino acid residues; the encoding gene has a nucleotide sequence shown as in SEQ ID No. 1 or a nucleotide sequence that hybridizes with the nucleotide sequence shown in SEQ ID No. 1 under strict conditions. After the encoding gene GLW2 is recombined to an expression vector and converted to rice, overexpression of GLW2 can increase rice grain length and width and increase rice yield.
Description
Technical field
The invention belongs to plant genetic engineering and plant breeding, particularly relate to a kind of rice grain shape associated protein GLW2 and encoding gene thereof and application.
Background technology
Oryza sativa L., as important cereal crops, has important effect in the Food Security of China, and improving constantly Oryza glutinosa yield per unit area is matters vital to national well-being and the people's livelihood. The character such as endosperm structure, grain of rice outward appearance, mass of 1000 kernel, setting percentage, grain number per spike are all had tremendous influence by grain type, are not only the important indicator of rice quality, are also the key factors of yield increase simultaneously. With the development of gene mapping colony, and the further exploitation of New molecular marker, there is more Oryza sativa L. key character gene to be positioned and clone successively. Research about these new gene greatly strengthens people's cognition to rice grain character inheritance characteristic and molecule mechanism, and the Appropriate application of these genes instructs and promoted the increase of rice yield and the raising of quality simultaneously.
So far, GrameneQTL (http://www.gramene.org/QTL) data base has included cultivated rice QTL8646, wherein Correlated Yield Characters QTL2064, relevant QTL219 of grain type, comprise 26, grain length site of control, grain is wide, grain is thick in control, 31,3,9 respectively, length-width ratio site, controls 22, grain weight site. Grain principal characteristic shape QTL is less likely to owing to grain weight gene prediction programs is relatively strong, inherited character relative complex, and what character identification relative difficulty caused. The relevant QTL of grain type is mainly distributed on the 1st, the 2nd, the 3rd and the 5th chromosome. In different positioning results, the QTL of various trait collects and is likely at same chromosome segment, and this can represent gene linkage or one because of the situation of multiple-effect, and the multiple character of the grain type in colony that explains synchronizes the phenomenon occurred. On the other hand, the QTL of same character is likely to and navigates on coloured differently body, and this then explains a source for type quantitative trait from Chromosome level.
In recent years, under the actively effort of the whole world particularly Chinese Scientists, the clone of rice grain shape and yield traits related gene achieves considerable progress, but the complexity due to quantitative trait QTL clone, result in the rice grain shape cloned now and yield traits related gene is also little, the mechanism regulating and controlling grain type in Oryza sativa L. is appointed so unclear, imperfection.Therefore, the clone of rice grain grain type related gene GLW2 and functional study, the molecular mechanism and its application in cross-breeding regulating and controlling seed grain type in clear and definite Oryza sativa L. is had great importance. .
Summary of the invention
The primary and foremost purpose of the present invention is in that to provide a kind of rice grain shape associated protein GLW2.
It is still another object of the present invention to provide the encoding gene of above-mentioned rice grain shape associated protein GLW2.
Another object of the present invention is to the application providing above-mentioned rice grain shape associated protein GLW2 or encoding gene in controlling rice grain grain type (length and width), improving rice grain weight and yield.
The present invention is realized in, application map-based cloning obtains the gene that a seed grain type is relevant from rice material 307R, the sudden change of this gene causes that rice grain grain length, grain are wide and dramatically increases, thus causing that seed grain heavily increases, No. 2 chromosomes grain length grain width gene (GLW2) of called after.
Thus, the invention provides a kind of rice grain shape associated protein GLW2, there is the aminoacid sequence as shown in SEQIDNO.2.
Additionally, present invention additionally comprises polypeptide, it is possible to be recombinant polypeptide, natural polypeptides, synthesis polypeptide, it is preferred that recombinant polypeptide. The polypeptide of the present invention can be native purified product, or the product of chemosynthesis, or uses recombinant technique to produce from protokaryon or eucaryon host (such as, antibacterial, yeast, higher plant, insecticide and mammalian cell). According to the host used by recombinant production scheme, the polypeptide of the present invention can be glycosylated, can be maybe nonglycosylated. The polypeptide of the present invention may also include or not include the methionine residues initiateed.
Present invention additionally comprises the fragment of GLW2 albumen, derivant and analog. herein, term " fragment ", " derivant " refer to the polypeptide of biological function that the GLW2 albumen being kept substantially the present invention is identical or activity with " analog ". the polypeptide fragment of the present invention, derivant or the like can be the polypeptide that (1) has one or more conservative or non-conservative amino acid residue (preferred Conservative amino acid residue) to be replaced, and the amino acid residue of such replacement can may not be and be encoded by genetic code, or (2) have the polypeptide of substituted radical in one or more amino acid residues, or (3) mature polypeptide (such as extends the compound of polypeptide half-life with another compound, such as Polyethylene Glycol) merge the polypeptide formed, or the polypeptide that (4) additional aminoacid sequence is fused to this peptide sequence and is formed is (such as targeting sequencing or secretion sequence or the sequence or the protein sequence that are used for this polypeptide of purification, or fusion protein). according to definition herein, these fragments, derivant and analog belong to the scope of well known to a person skilled in the art.
In the present invention, GLW2 albumen refers to the polypeptide with SEQIDNO.2 sequence. Present invention additionally comprises albumen identical function, the SEQIDNO.2 sequence variant form with SEQIDNO.2 sequence. These variant forms include (but being not limited to): several (are generally 1��50, preferably 1��30, more preferably 1��20,1��10 or less 1��8 or 1��5 best) amino acid whose disappearance, insertion and/or replacement, and (it is generally within 20 at C-terminal and/or N-terminal interpolation one or several, it is preferably within 10, is more preferably within 5) aminoacid. Such as, in this area, when replacing with the aminoacid that performance is similar, the function of typically not change protein. Again such as, one or several general designation are added without the function changing protein at C-terminal and/or N-terminal.Also include active fragment and the reactive derivative of GLW2 albumen.
The variant form of polypeptide includes: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutants, can with the albumen coded by the DNA of GLW2 encoding histone DNA hybridization and utilize the polypeptide or albumen that the antiserum of GLW2 albumen obtains under high or low stringency conditions. Described homologous sequence refers to have have at least 50% with SEQIDNO.2 sequence, it is preferred that at least 60%, 70%, 80%, more preferably at least 85%, 90%, and the polypeptide of 95% homogeny. Present invention also offers other polypeptide, as comprised the fusion protein of GLW2 albumen or its fragment. Except the polypeptide of almost total length, present invention includes the soluble fragments of GLW2 albumen. Generally, this fragment has at least about 20 the continuous print aminoacid of GLW2 albumen, typically at least about 30 continuous amino acids, it is preferred that at least about 50 continuous amino acids, more preferably at least about 100 continuous print aminoacid, best at least about 150 continuous amino acids.
The present invention also provides for the analog of GLW2 albumen or polypeptide. The difference of these analog and natural GLW2 albumen can be the difference on aminoacid sequence, it is also possible to is do not affect the difference on the modified forms of sequence, or haves both at the same time. These polypeptide include natural or induction genetic variant. Induction variant can be obtained by various technology, such as the random mutagenesis produced by radiating or be exposed to mutagenic agent, it is also possible to by site-directed mutagenesis or other known molecular biological technology. Analog also includes having the analog being different from natural L-amino acid residue (such as D-aminoacid), and has non-naturally-occurring or synthesis the analog of aminoacid (such as beta-amino acids). Should be understood that the polypeptide of the present invention is not limited to the above-mentioned representative polypeptide enumerated.
(generally the not changing primary structure) form of modification includes: the chemically derived form such as acetylation or carboxylated of inner or in vitro polypeptide. Modify and also include glycosylation. Modified forms also includes the sequence with phosphorylated amino acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine). Also include being modified thus improve its anti-Proteolytic enzyme performance or optimizing the polypeptide of solubility property.
Also include GLW2 albumen conservative variation's albumen (polypeptide) in the present invention, compared with the aminoacid sequence of SEQIDNO.2, there are at most 20, preferably at most 10, more preferably at most 5, at most 3 aminoacid is substituted by the aminoacid that character is similar or close and the polypeptide that formed best, and retains the function identical with the albumen of SEQIDNO.2 sequence.
Further, the invention provides the encoding gene of above-mentioned rice grain shape associated protein GLW2, this encoding gene is have the nucleotide sequence as shown in SEQIDNO.1; Or, under strict conditions with the nucleotide sequence of the nucleotide sequence hybridization shown in SEQIDNO.1. That is, present invention also offers the polynucleotide sequence of code book invention GLW2 albumen or its conservative variation's polypeptide.
The polynucleotide (gene) of the present invention can be DNA form or rna form. DNA form includes the DNA of cDNA, genomic DNA or synthetic. DNA can be strand or double-strand. DNA can be coding strand or noncoding strand. The coding region sequence of encoding mature polypeptide can the variant of or degeneracy identical with the coding region sequence shown in SEQIDNO.2.As used herein, " variant of degeneracy " refers in the present invention and encodes the protein with SEQIDNO.2, but nucleotide sequence differentiated with the coding region sequence shown in SEQIDNO.2.
The polynucleotide of the mature polypeptide of coding SEQIDNO.2 include: the coded sequence of an encoding mature polypeptide; The coded sequence of mature polypeptide and various additional coding sequence; The coded sequence (with optional additional coding sequence) of mature polypeptide and non-coding sequence.
The invention still further relates to the variant of above-mentioned polynucleotide, its coding and the present invention have polypeptide or the fragment of polypeptide, the sum analogous to general Dedekind sum of identical aminoacid sequence. The variant that the variant of these polynucleotide can be the allelic variant of natural generation or non-natural occurs. These nucleotide variants include replacing variant, Deletion variants and insertion variant. As known in the art, allelic variant is the alternative forms of polynucleotide, and he is probably the replacement of one or more nucleotide, disappearance or insertion, but will not from the function of the polypeptide substantially changing its coding.
The invention still further relates to and have at least 50% between above-mentioned sequence hybridization and two sequences, it is preferred that at least 70%, further preferably at least 80%, the more preferably polynucleotide of at least 90% homogeny. The present invention be more particularly directed to polynucleotide interfertile with polynucleotide of the present invention under strict conditions. In the present invention, " stringent condition " refers to: (1) hybridization under relatively low ionic strength and higher temperature and eluting, such as 0.2 �� SSC, 0.1%SDS, 60 DEG C; Or added with denaturant during (2) hybridization, such as 50% (V/V) Methanamide, 0.1% calf serum/0.1Ficoll, 42 DEG C etc.; Or (3) only homogeny between two sequences is at least more than 70%, it is preferred that more than at least 80%, more preferably more than 90%, just hybridize when being more preferably more than 95%. Further, the polypeptide of interfertile polynucleotide encoding and the mature polypeptide shown in SEQIDNO.2 have identical biological function and activity.
The invention still further relates to and the nucleic acid fragment of above-mentioned sequence hybridization. As used herein, the length of " nucleic acid fragment " at least contains 15 nucleotide, is preferably at least 30 nucleotide, is at least 50 nucleotide better, it is preferred to more than at least 100 nucleotide. Adjust amplification technique (such as PCR) that fragment can be used for adjusting to determine and/or the polynucleotide of separately coded GLW2 albumen.
The GLW2 protein nucleotides full length sequence of the present invention or its fragment generally can use the method for pcr amplification method, recombination method or synthetic to obtain. For pcr amplification method, can according to relevant nucleotide sequence disclosed by the invention, especially open reading frame designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by conventional method well known by persons skilled in the art as template, amplification and obtain relevant sequence. When sequence is longer, it is often necessary to perform twice at or repeatedly pcr amplification, then again the fragment that each time amplifies is stitched together in the correct sequence.
Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method. This is usually cloned into carrier, then proceeds to cell, then passes through conventional method separation from the host cell of increment sum and obtains relevant sequence.
Further, it is also possible to utilize the method for synthetic to synthesize relevant sequence, when especially fragment length is shorter.Generally, first synthesize multiple small fragment, then can obtain the fragment that sequence is very long being attached.
At present, it is already possible to be entirely through chemosynthesis to obtain the DNA sequence of code book invention albumen (or its fragment, or derivatives thereof). Then this DNA sequence can be introduced in various existing DNA moleculars (or such as carrier) as known in the art and cell. Additionally, also sudden change can be introduced in protein sequence of the present invention with crossing chemosynthesis.
Further, present invention also offers the expression vector of encoding gene containing above-mentioned rice grain shape associated protein GLW2, and the carrier or GLW2 albumen coded sequence by the present invention produces host cell and the method through recombinant technique generation polypeptide of the present invention through genetic engineering. By conventional DNA recombinant technique, the polynucleotide sequence of the available present invention is expressed or produces the GLW2 albumen of restructuring. Generally there are following steps:
The polynucleotide encoding GLW2 albumen obtained by the present invention or variant, or convert or suitable host cell of transduceing with the recombinant expression carrier of these polynucleotide;
Suitable culture medium is cultivated host cell;
Separation, protein purification from culture medium or cell.
In the present invention, GLW2 protein polynucleotide can be plugged in recombinant expression carrier. Term " recombinant expression carrier " refers to bacterial plasmid well known in the art, phage, yeast plasmid, plant cell virus, mammalian cell virus or other carriers. In a word, as long as can replicate in host and stable, any plasmid and carrier can use. One key character of expression vector is to usually contain origin of replication, promoter, marker gene and translation to control element.
Method well-known to those having ordinary skill in the art can be used for building containing GLW2 protein coding DNA sequence and the suitable expression vector transcribing, translating control signal. These methods include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc. Described DNA sequence can be effectively connected in promoter suitable in expression vector, to instruct the synthesis of mRNA. Expression vector also includes ribosome binding site and the transcription terminator of translation initiation.
In addition, expression vector preferably comprises one or more selected marker, to provide the phenotypic character of the host cell for selecting conversion, such as eukaryotic cell cultivation dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP), or for colibacillary kanamycin or amicillin resistance.
Comprise above-mentioned suitable DNA sequence and suitable promoter or control sequence carrier, it is possible to for converting suitable host cell, allowing it to marking protein.
Host cell can be prokaryotic cell, such as bacterial cell; Or the eukaryotic cell such as low, such as yeast cells; Or higher eucaryotic cells, such as plant cell. Representational example has: escherichia coli, streptomyces, Agrobacterium; Eukaryotic cell is yeast such as; Plant cell etc.
Further, present invention also offers the purposes of above-mentioned rice grain shape associated protein GLW2 and encoding gene thereof, specifically include:
GLW2 gene map based cloning, as shown in Figure 1, by IR24 is background GLW2 NIL colony, GLW2 is pin-pointed within the scope of No. 2 chromosome long arm end 15.3K of Oryza sativa L., find again through to the order-checking comparison in this region, there is true base mutation (TC-AA) in one of them gene, is GLW2 by this unnamed gene;
Control rice grain grain length, grain are wide;
The method improving rice yield.
The present invention can pass through to regulate expression or the activity of GLW2 gene in described plant, and the adjustment seed grain length of Oryza sativa L., grain are wide, thus increasing seed grain weight. Promote or suppress the expression activity of GLW2 to depend primarily on the character of the required improvement of Oryza sativa L. and determine. When needing the seed grain weight increasing Oryza sativa L., it is possible to realized by the expression or activity improving GLW2 gene in described Oryza sativa L.; When needing to reduce the seed grain weight of Oryza sativa L., it is possible to realized by the expression or activity reducing GLW2 gene in described plant.
The present invention is by NIL NIL-GS2527R��NIL-GS2MH63And corresponding parent 527R, MH63 hybridize with sterile line 640A, 106A respectively, by hybrid F1For 640A/MH63,640A/NIL-GS2MH63��106A/MH63��106A/NIL-GS2MH63��640A/527R��640A/NIL-GS2527RGS2 yield potentiality is estimated by yield traits investigation, as shown in table 1 below and table 2.
All NIL groups join that the seed grain length of offspring's strain, grain be wide, mass of 1000 kernel and spike length pole dramatically increase; Its grain number per spike and tiller number are without significant change; 640A/NIL-GS2MH63And 106A/NIL-GS2MH63Group join in offspring, plant height has extremely to be increased significantly, and 640A/NIL-GS2527RGroup joins offspring's plant height without significant change, it is presumed that causing change of height is that the factors such as Combining Ability of Different Parental Varieties cause, is not exposed to the impact of GS2; All NIL groups join offspring's strain single plant yield and every square metre of yield dramatically increases, wherein in every square metre of yield, and 106A/NIL-GS2MH63Group join offspring and increase production 13.74%, 640A/NIL-GS2527RGroup is joined offspring and is increased production 17.85%, 640A/NIL-GS2MH63Group is joined offspring and is increased production 28.00%. Investigate and analyse us by yield traits it is found that in different parents offsprings the yield potential of GS2 different, its amount of increase in production is between 13.74%-28.00%. Thus it is contemplated that, on the basis of reasonable Selection parent combining ability, GS2 has higher yield potential.
Table 1 NIL cross combination yield traits
Table 2 NIL cross combination yield traits (continued 1)
The method of the expression increasing GLW2 gene is well known in the art. Such as, can pass through to proceed to the expression vector carrying GLW2 encoding gene and make plant overexpression GLW2; Maybe can be driven thus strengthening GLW2 gene or its homogenic expression by strong promoter; Or the expression of this GLW2 gene is strengthened by enhancer (such as Oryza sativa L. waxy gene First Intron, Actin gene First Intron). Strong promoter suitable in the inventive method includes but not limited to: 35S promoter, Actin1, Actin2 promoter of Oryza sativa L., Semen Maydis Ubi promoter etc.
The method suppressing GLW2 gene expression is also well known in the art, for instance, antisense or RNA interference (RNAi) can be passed through or clpp gene out realizes.
A kind of optimal way as the present invention, it is thus achieved that the method for the plant of GLW2 gene high expression is as follows:
Thering is provided the Agrobacterium carrying expression vector, described expression vector contains the coded sequence of described GLW2 albumen;
Plant cell, tissue or organ are contacted with the Agrobacterium in step (1), so that described GLW2 albumen coded sequence proceeds to plant cell, and is incorporated on the chromosome of plant cell;
Select and proceed to the plant cell of described GLW2 albumen coded sequence, tissue or organ;
Plant cell in step (3), tissue or neomorph are become plant.
Wherein, any suitable conventional means can be adopted, implement the method including reagent, temperature, pressure condition.
According to a specific embodiment of the present invention, it is provided that a kind of GLW2 albumen, open reading frame (ORF) sequence of its total length, such as shown in SEQIDNO.1, encodes one containing 394 amino acid whose protein (SEQIDNO.2).
In the specific embodiment of the present invention, construct GLW2 gene overexpression carrier PHB-GLW2 and GLW2 gene knockout carrier BGK03-GLW2, by transgene method, normal wild type gene GLW2 is imported to rice material Japan fine in can make its seed grain length, increase grain wide; The knockout carrier of GLW2 is imported in GLW2 NIL plant, seed grain length can be reduced, to increase grain wide.
Accompanying drawing explanation
Fig. 1 is GLW2 gene clone site figure of the present invention;
BGK03 carrier structure schematic diagram in Fig. 2 embodiment of the present invention;
Fig. 3 is Oryza sativa L. plant seed phenotype after GLW2 overexpression in the embodiment of the present invention;
Fig. 4 is Oryza sativa L. GLW2 expression testing result after GLW2 overexpression in the embodiment of the present invention;
Fig. 5 is Oryza sativa L. strain seed mass of 1000 kernel statistics after GLW2 overexpression in the embodiment of the present invention;
Fig. 6 is Oryza sativa L. strain seed grain length statistics after GLW2 overexpression in the embodiment of the present invention;
Fig. 7 is Oryza sativa L. wide statistics of strain seed grain after GLW2 overexpression in the embodiment of the present invention;
Fig. 8 is Oryza sativa L. plant seed phenotype after GLW2 knocks out in the embodiment of the present invention;
Fig. 9 is Oryza sativa L. plant seed mass of 1000 kernel statistics after GLW2 knocks out in the embodiment of the present invention;
Figure 10 is Oryza sativa L. plant seed grain length statistics after GLW2 knocks out in the embodiment of the present invention;
Figure 11 is Oryza sativa L. wide statistics of plant seed grain after GLW2 knocks out in the embodiment of the present invention;
Figure 12 is the comparison diagram of the phenotype of GLW2 gene mutation kind and normal water rice varieties phenotype in the embodiment of the present invention.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated. Should be appreciated that specific embodiment described herein is only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1GLW2 trans-genetic hybrid rice Transgenic studies
1, GLW2 gene overexpression expression vector establishment
The present embodiment adopts expression vector PHB (Mao etc., 2005, PNAS102:12270-12275, commercially available) as the carrier of Transgenic Rice. This vector encoded one bacterial origin of replication (ori), kalamycin resistance gene (Kanr), hygromycin gene (Hygr), herbicide resistance gene (Barr), 2 �� 35S promoter, NOS gene-end signal sequence and for connecting the multiple clone site (MCS) of purpose fragment. GLW2 gene is can be inserted into or its fragment is built into transgenic plasmid at restriction endonuclease sites.
With the RNA of normal wild type IR24 kind for template, synthesize the first chain cDNA, with 5 ' and the 3 ' oligonucleotide held of this GLW2 gene ORF sequence as PCR primer (SEQIDNO.3 and SEQIDNO.4), expand with high-fidelity enzyme, it is thus achieved that the full-length cDNA amplification product of the GLW2 gene of about 1185bp. This amplified production is recombinated in carrier pHB by recombinant clone method, and recon is carried out sequence verification. The transition plasmid vector of this restructuring is called PHB-GLW2. Wherein the expression of GLW2 gene is driven by its own promoter.
The oligonucleotide sequence 3 that 5 ' hold is (SEQIDNO.3):
5��-gtctctctctcaagcttggatccATGGCGATGCCGTATGCCTCC-3��
The oligonucleotide sequence 4 that 3 ' hold is (SEQIDNO.4):
5��-tctagaggatcaattcgagctcTCAGTCACCATTAGTTGATCGAG-3��
2, GLW2 knockout carrier builds
Utilizing CRISPR/Cas9 system constructing carrier, container name is BGK03 (hundred lattice biotech companies buy), and carrier structure schematic diagram is as shown in Figure 1. Adopt Oryza sativa L. U6 promoter, it is possible to efficiently for monocotyledon, particularly Oryza sativa L.; Adopt reinforced Semen Maydis UBI promoter, high efficient expression Cas9 albumen;CaMV35S promoter expression hygromycin gene.
First design and generate gRNA target sequence. Recognition sequence is typically chosen 19bp, such as select target sequence GCTCATATACAAGTACCTGGTGG (TGG of underscore part is PAM sequence), then target sequence (SEQIDNO.5) and (SEQIDNO.6) are designed, the primer of synthesis is dissolved in water to 10 ��Ms, after mixing by following reaction system, 95 DEG C are heated 3 minutes, are then slowly dropped to 20 DEG C (recommending to adopt PCR instrument) with about 0.2 DEG C/sec. The primer dimer generated in finally upper step being reacted under the effect of enzyme is built into carrier B GK03, and the container name built is BGK03-GLW2.
The oligonucleotide sequence 5 that 5 ' hold is (SEQIDNO.5):
5 '-TGTGTGCTCATATACAAGTACCTGG-3 ';
The oligonucleotide sequence 6 that 3 ' hold is (SEQIDNO.6):
5 '-AAACCCAGGTACTTGTATATGAGCA-3 ';
3, GLW2 gene transformation rice test
Upper PHB-GLW2 and BGK03-GLW2 is imported Agrobacterium EHA105 by freeze-thaw method. Agrobacterium EHA105 flat board (4 DEG C of preservations) picking list bacterium colony is in YEP fluid medium (each 50mg/L of Km and Rif), 28 DEG C of shaken cultivation 12��18h, then take 1��5mL bacterium solution and receive in 100mLYEP fluid medium (containing 100 ��m of ol/L of acetosyringone), survey OD value after shaken cultivation 4h rare to respective concentration (OD=0.5). By fresh bacterium solution in 8000rpm, 4 DEG C, 5min collects thalline, and is resuspended in 1/3rd AAM culture medium mentioned. Add and the sterilizing triangular flask being placed with eugonic embryo callus soaks 25min, then dry up surface bacterium solution, callus is transferred in co-culturing on base, 28 DEG C of light culture 2��3d. Co-culturing callus after sterilized water and the rinsed with sterile water containing Cef500mg/L, blow about 4h on the table, transfer 28 DEG C of light culture 5��7d in pre-culture. Pre-incubated callus is transferred in the screening culture medium containing Hyg and Cef, continues cultivation 3��4 weeks, then resistant calli is transferred in only screening culture medium containing Hyg, screen 1��2 time. Take resistant calli and transfer on division culture medium, 28 DEG C of illumination differentiation. Differentiation seedling is transferred in root media, 28 DEG C of illumination cultivation 3��4 weeks, and open culturing seedling exercising about 7d is finally transplanted to land for growing field crops.
The Transplantation of Regenerated Plantlets obtained survive after with herbicide or hygromycin selection transformed plant; Positive plant extracts blade STb gene, identifies transformed plant further through PCR. By transgenic T1 generation investigation fertility phenotype, verify GLW2 gene function.
Embodiment 2GLW2 gene function analysis
Method as described in Example 1 obtains the transfer-gen plant of Oryza sativa L. GLW2 overexpression, it is divided into 2 groups, it is respectively designated as OE1-2, OE2-3, and Normal group control, with transgenic T1 for plant observe rice grain phenotype, result as shown in figure 3 to figure 7, wherein, in Fig. 4��7, * * is that notable NS is comparison unintentionally in 0.01 level).
It can be seen that turn, the plant seed grain length of GLW2 overexpression gene, grain be wide and mass of 1000 kernel has relatively compareed and dramatically increased. Show that GLW2 overexpression can increase seed grain length, grain wide, and then cause that mass of 1000 kernel increases.
Method as described in Example 1 obtains the Oryza sativa L. GLW2 transfer-gen plant knocked out, it is divided into 4 groups, it is respectively designated as KO1-1, KO1-2, KO1-5 and KO2-3, WT is wild type material, rice grain phenotype is observed for plant with transgenic T1, wherein, GLW2 knocks out plant to knock out site as shown in table 3 below:
Table 3GLW2 knocks out plant and knocks out Locus Analysis in Shoots
Result is such as shown in Fig. 8��11, and in Fig. 8, NIL-527R is the NIL of background, and in Fig. 9��11, * * is notable in 0.01 level.From Fig. 8��11 it can be seen that the seed grain length of plant after GLW2 gene editing, grain are wide and mass of 1000 kernel has relatively compareed and significantly reduced. Show that GLW2 knocks out that to reduce seed grain length, grain wide, and then cause that mass of 1000 kernel reduces.
As can be seen from the above analysis, it is wide that GLW2 can control rice grain grain length, grain, and then affects rice yield, raises this gene expression and is conducive to plant seed grain length, the wide increase of grain, and lowering this gene expression, then to reduce the seed grain length of plant, grain wide.
The phenotype (GLW2 position natural mutation material) of embodiment 3GLW2 gene mutation
Rice varieties 307R (being stored in resource center of Inst. of Paddy Rice, Sichuan Agriculture Univ.) after GLW2 gene mutation, show through table analysis and statistical analysis, 307R seed is compared to existing normal water rice varieties 527R, IR24 and M63, and its grain length, grain be wide and mass of 1000 kernel relatively other rice materials dramatically increase (Figure 12 and Biao 4).
Table 4 normal water rice varieties and 307R complete ripeness seed species test Data-Statistics
Compared to the shortcoming and defect of prior art, the method have the advantages that rice grain shape of the present invention controls the technological approaches that the seed grain type regulation and control that gene (GLW2) is the plants such as Oryza sativa L. provide new; GLW2 gene is with a wide range of applications in removing transgene component and affecting; This gene can significantly improve rice yield, is with a wide range of applications in rice heterosis utilization.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all any amendment, equivalent replacement and improvement etc. made within the spirit and principles in the present invention, should be included within protection scope of the present invention.
Claims (10)
1. a rice grain shape associated protein GLW2, it is characterised in that there is the aminoacid sequence as shown in SEQIDNO.2; Or for the aminoacid sequence with equal function that the aminoacid sequence shown in SEQIDNO.2 is formed through the replacement of one or more amino acid residues, disappearance or interpolation.
2. the encoding gene of the rice grain shape associated protein GLW2 described in claim 1, it is characterised in that the nucleotides sequence of this encoding gene is classified as: have the nucleotide sequence as shown in SEQIDNO.1; Or, under strict conditions with the nucleotide sequence of the nucleotide sequence hybridization shown in SEQIDNO.1.
3. the encoding gene of rice grain shape associated protein GLW2 as claimed in claim 2, it is characterised in that described stringent condition is 0.2 �� SSC, 0.1%SDS, 60 DEG C; Or 50% (V/V) Methanamide, 0.1% calf serum/0.1Ficoll, 42 DEG C.
4. contain the expression vector of encoding gene described in Claims 2 or 3.
5. expression vector as claimed in claim 4, it is characterised in that described expression vector includes PHB, BGK03.
6. contain the encoding gene described in Claims 2 or 3 or the host cell containing the expression vector described in claim 4 or 5.
7. the rice grain shape associated protein GLW2 described in claim 1 is in the application controlling the wide improvement of rice grain grain length, grain, improve in rice yield.
8. the encoding gene described in Claims 2 or 3 is in the application controlling the wide improvement of rice grain grain length, grain, improve in rice yield.
9. the application in preparing transfer-gen plant of the rice grain shape associated protein GLW2 described in claim 1.
10. the application in preparing transfer-gen plant of the encoding gene described in Claims 2 or 3.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011146A (en) * | 2016-06-13 | 2016-10-12 | 中国农业科学院作物科学研究所 | Application of OsMADS47 gene to regulation of rice grain form |
| CN106119280A (en) * | 2016-07-14 | 2016-11-16 | 湖南新春农业生物高科技有限公司 | The albumen OsJGL2 relevant to rice grain length and encoding gene thereof and application |
| CN107298702A (en) * | 2017-08-09 | 2017-10-27 | 四川农业大学 | A kind of rice grain shape GAP-associated protein GAP and its encoding gene |
| CN109422803A (en) * | 2017-08-31 | 2019-03-05 | 中国科学院上海生命科学研究院 | Adjust gene and its application of plant particle shape, mass of 1000 kernel and grain number per spike |
| WO2019158911A1 (en) * | 2018-02-14 | 2019-08-22 | Institute Of Genetics And Developmental Biology Chinese Academy Of Sciences | Methods of increasing nutrient use efficiency |
| CN112609018A (en) * | 2020-12-11 | 2021-04-06 | 华智生物技术有限公司 | SNP molecular marker of rice grain type related gene GLW2 and application thereof |
| CN115725597A (en) * | 2022-07-05 | 2023-03-03 | 四川农业大学 | Rice grain width and weight regulation gene DWG1 and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102329806A (en) * | 2011-09-30 | 2012-01-25 | 中国科学院遗传与发育生物学研究所 | Gene for controlling rice grain width, grain weight and yield and application thereof |
| CN103451228A (en) * | 2013-09-17 | 2013-12-18 | 淮阴师范学院 | Method for regulating size and grain weight of rice seeds |
| CN104745599A (en) * | 2013-12-31 | 2015-07-01 | 华中农业大学 | Rice granule shape gene qSS7 as well as preparation method and application |
-
2016
- 2016-03-08 CN CN201610128841.XA patent/CN105646684B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102329806A (en) * | 2011-09-30 | 2012-01-25 | 中国科学院遗传与发育生物学研究所 | Gene for controlling rice grain width, grain weight and yield and application thereof |
| CN103451228A (en) * | 2013-09-17 | 2013-12-18 | 淮阴师范学院 | Method for regulating size and grain weight of rice seeds |
| CN104745599A (en) * | 2013-12-31 | 2015-07-01 | 华中农业大学 | Rice granule shape gene qSS7 as well as preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| NONE: "Accession NO.: XP_015624297.1，PREDICTED: growth-regulating factor 4 [Oryza sativa Japonica Group]", 《GENBANK DATABASE》 * |
| 杨联松等: "水稻粒形与稻米品质间相关性研究进展", 《安徽农业科学》 * |
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|---|---|---|---|---|
| CN106011146A (en) * | 2016-06-13 | 2016-10-12 | 中国农业科学院作物科学研究所 | Application of OsMADS47 gene to regulation of rice grain form |
| CN106011146B (en) * | 2016-06-13 | 2019-06-21 | 中国农业科学院作物科学研究所 | Application of the OsMADS47 gene in adjusting and controlling rice grain shape |
| CN106119280A (en) * | 2016-07-14 | 2016-11-16 | 湖南新春农业生物高科技有限公司 | The albumen OsJGL2 relevant to rice grain length and encoding gene thereof and application |
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| CN107298702B (en) * | 2017-08-09 | 2020-10-23 | 四川农业大学 | Rice grain type related protein and coding gene thereof |
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| WO2019158911A1 (en) * | 2018-02-14 | 2019-08-22 | Institute Of Genetics And Developmental Biology Chinese Academy Of Sciences | Methods of increasing nutrient use efficiency |
| CN111818794A (en) * | 2018-02-14 | 2020-10-23 | 中国科学院遗传与发育生物学研究所 | Method for increasing nutrient utilization efficiency |
| US11873499B2 (en) | 2018-02-14 | 2024-01-16 | Institute Of Genetics And Developmental Biology Chinese Academy Of Sciences | Methods of increasing nutrient use efficiency |
| CN112609018A (en) * | 2020-12-11 | 2021-04-06 | 华智生物技术有限公司 | SNP molecular marker of rice grain type related gene GLW2 and application thereof |
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